Biodegradable hydrogel matrices for the controlled release of pharmacologically active agents

ABSTRACT

A biodegradable hydrogel matrix is provided, useful for the controlled release of pharmacologically active agents. The matrix is formed by cross-linking a proteinaceous component and a polysaccharide or mucopolysaccharide, and then loading a selected drug therein. By varying temperature, ionic strength, and the composition of the hydrogel matrix, one can control degradation kinetics, the degree of uptake of a particular pharmacologically active agent, and the overall times release profile.

DESCRIPTION

1. Technical Field

This invention relates generally to drug delivery systems, and moreparticularly relates to biodegradable hydrogel matrices useful in thecontrolled release of pharmacologically active agents.

2. Background

The last decade has seen rapid development in the area of drug delivery.In particular, a number of drug delivery systems have been developed toeffect the controlled release of pharmacologically active agents. For ageneral overview of the art, reference may be had, inter alia, to R.Baker, Controlled Release of Biologically Active Agents, New York: JohnWiley & Sons, 1987.

One area of research has been in the use of "hydrogels", orwater-swellable polymeric matrices, in drug delivery systems. See, forexample, P.I. Lee, J. Controlled Release 2:277-288 (1985). Hydrogels arenetwork polymers which can absorb a substantial amount of water to formelastic gels. The release of pharmacologically active agents "loaded"into such gels typically involves both absorption of water anddesorption of the agent via a swelling-controlled diffusion mechanism.

A significant drawback in the use of hydrogels, however, and one thathas substantially hindered the use of hydrogels in drug deliverysystems, is that such formulations are generally not biodegradable.Thus, drug delivery devices formulated with hydrogels typically have tobe removed after subcutaneous or intramuscular application or cannot beused at all if direct introduction into the blood stream is necessary.Thus, it would be advantageous to use hydrogels which could be degradedafter application in the body without causing toxic or other adversereactions.

Only a few types of biodegradable hydrogels have been described. Thesehave been based on proteins (e.g., using albumin microspheres, asdescribed in S.S. Davis et al., J. Controlled Release 4:293-303 (1987))or on poly(αamino acids), as described in H.R. Dickinson et al., J.Biomed. Mater. Res. 15: 577-589 and 591-603 (1981)). Even theseformulations, however, have proved problematic with regard tobiocompatibility.

Collagen matrices, including collagen-mucopolysaccharide matrices, havebeen prepared and used for wound healing applications and in laminatedmembranes useful as synthetic skin. See, e.g., U.S. Pat. Nos. 4,060,081,4,280,954 and 4,418,691 to Yannas et al. and 4,485,096 to Bell. Thesecollagen matrices, however, would have limited if any utility in drugdelivery as they are not "blood compatible". That is, the properties ofthese matrices that enable wound healing--e.g., by facilitatingclotting--teach against their use in drug delivery systems.

The inventor herein has now discovered a biodegradable hydrogel whichhas significantly enhanced biocompatibility in that (1) bloodcompatibility is substantially improved, (2) immunogenicity isminimized, and (3) the hydrogel is enzymatically degraded to endogenous,nontoxic compounds. The process for making the novel hydrogel representsa further advance over the art in that, during synthesis, one cancarefully control factors such as hydrophilicity, charge and degree ofcross-linking. By varying the composition of the hydrogel as it is made,one can control the uptake of a particular drug, the degradationkinetics of the hydrogel formulation and the overall timed-releaseprofile.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates the preparation of a biodegradablehydrogel as described in Example 1.

FIGS. 2 and 3 schematically illustrate the preparation ofalbumin-heparin microspheres as described in Example 2.

FIGS. 4 and 5 are scanning electron micrograph is of "chemicallystabilized" microspheres prepared as described in Example 2.

FIGS. 6 through 9 illustrate in graph form the swelling behavior ofalbumin-heparin microspheres in buffer solution.

FIG. 10 illustrates in graph form the release of protein from an albuminhydrogel, as described in Example 4.

FIGS. 11 and 12 illustrate the release profile of lysozyme-loadedalbumin-heparin gels as described in Example 5.

DISCLOSURE OF THE INVENTION

A drug delivery system comprising:

(a) a biodegradable hydrogel matrix comprising a protein, apolysaccharide, and a cross-linking agent providing network linkagetherebetween, wherein the weight ratio of polysaccharide to protein inthe matrix is in the range of about 10:90 to 90:10; and

(b) a drug contained within the matrix.

The invention also encompasses a method of making such a drug deliverysystem, comprising dissolving the aforementioned components in anaqueous medium, cross-linking the components to provide athree-dimensional network, and loading a selected drug, in solution orin liquid form, into the matrix. The composition of the hydrogel formedmay be varied during synthesis so as to alter hydrophilicity, charge anddegree of cross-linking.

As the systems of the present invention are blood- andtissue-compatible, they may be used to deliver a variety of drugsaccording to any number of modes of administration, e.g., oral,parenteral, or the like.

As noted above, a primary advantage of the novel hydrogels is in theirenhanced biocompatibility. The use of polysaccharides ormucopolysaccharides (especially heparins) in the formulations isbelieved to enhance blood compatibility and significantly reduceactivation of the complement system. Furthermore, as the polymericcomponents of the hydrogel are endogenous, the products of enzymaticdegradation are endogenous as well.

MODES FOR CARRYING OUT THE INVENTION

The drug delivery systems of the present invention are formed bycross-linking a polysaccharide or a mucopolysaccharide with a proteinand loading a drug, in solution or in liquid form, into the hydrogelmatrices so provided. The hydrogel matrices can be prepared usingdifferent ratios of polysaccharide or mucopolysaccharide to protein andcan be produced in various sizes and geometries. Upon incorporation ofthe selected drug as will be described, the hydrogel can be swollen tovarious extents depending on the composition of the gel as well as onpH, temperature, and the electrolyte concentration of the loadingmedium. This permits the incorporation of different types and classes ofdrugs, including low molecular weight drugs, peptides and proteins.After exposure of the drug-containing hydrogel to the physiologicalenvironment, i.e., to blood or tissue, drugs will be released gradually.The release rate, like the loading parameters, will depend on thecomposition of the gel, the degree of cross-linking, any surfacetreatment of the components (e.g., to increase or decrease theirhydrophilicity, charge, degradation kinetics), the type of drug used,and the geometry of the hydrogel body.

By "hydrogel" as used herein is meant a water-swellable,three-dimensional network of macromolecules held together by covalentcross-links. (These covalent cross-links are sometimes referred toherein as providing a "network linkage" within the macromolecularstructure.) Upon placement in an aqueous environment, these networksswell to the extent allowed by the degree of cross-linking.

By the term "pharmacologically active agent" or "drug" as used herein ismeant any chemical material or compound suitable for administrationwhich induces a desired systemic or local effect. In general, thisincludes therapeutic agents in all of the major therapeutic areas. By"effective" amount of a pharmacologically active agent or drug is meanta nontoxic but sufficient amount of a compound to provide the desiredsystemic or local effect.

By a drug or pharmacologically active agent in "liquid form", as usedherein, is intended a liquid drug, i.e., neat, or a drug dissolved ordispersed in a pharmacologically compatible carrier. By drug "containedwithin" a hydrogel matrix is meant a drug dispersed or dissolvedtherein.

By "protein", as used herein, is meant both full-length proteins andpolypeptide fragments, which in either case may be native, recombinantlyproduced, or chemically synthesized.

"Polysaccharides", as used herein, are intended to include bothpolysaccharides and mucopolysaccharides.

Examples of suitable polysaccharides include heparin, fractionatedheparins (e.g., on an AT-III column), heparan, heparan sulfate,chondroitin sulfate, and dextran. In general, the polysaccharides ormucopolysaccharides useful in forming the hydrogels of the invention arethose described in U.S. Pat. No. 4,060,081 to Yannas et al., citedsupra. Heparin or heparin analogs are preferable because the compoundsare strong anticoagulants and biodegradable by heparinases and amylases.In addition, using heparin or heparin analogs, i.e., compounds which arestructurally related to heparin and which provide the same or similardegree of biocompatibility, appears to reduce immunogenicity and,because the compounds are highly charged, aqueous swelling is high,facilitating drug loading and release.

The protein component of the hydrogel may be, as noted above, either afull-length protein or a polypeptide fragment. It may be in native form,recombinantly produced or chemically synthesized. This protein componentmay also be a mixture of full-length proteins and/or fragments.Typically, the protein is selected from the group consisting of albumin,casein, fibrinogen, γ-globulin, hemoglobin, ferritin and elastin. Thislist is intended to be illustrative and not in any way limiting. Forexample, the protein component of the hydrogel may also be a syntheticpolypeptide, e.g., a poly (α-amino acid) such as polyaspartic orpolyglutamic acid.

Albumin is preferred as the protein component of the matrix as it is anendogenous material biodegradable in blood by proteolytic enzymes, intissue by proteolytic enzymes associated with macrophage activity, andin different organs by phagocytosis, i.e., by the action of cells of thereticuloendothelial system (RES). Furthermore, albumin prevents adhesionof thrombocytes and is nontoxic and nonpyrogenic.

As noted above, a primary advantage of the invention lies in the factthat both the protein and polysaccharide components of the hydrogel areendogenous, biocompatible materials. This substantially reduces thelikelihood of immunogenicity and, further, ensures that the products ofbiodegradation are also biocompatible materials.

The weight ratio of polysaccharide or mucopolysaccharide to proteinwithin the hydrogel matrix is quite variable, and is typically withinthe range of about 10:90 to 90:10. More preferably, the range is about10:90 to 60:40. The selected ratio affects drug loading, degradationkinetics, and the overall timed release profile. Thus, by varying therelative amounts of the protein and polysaccharide components in thehydrogel, one can, to a large extent, control the aforementionedfactors.

In forming the novel hydrogels, one of several cross-linking methods maybe used:

(1) The polysaccharide or mucopolysaccharide and the protein may bedissolved in an aqueous medium, followed by addition of an amidebond-forming cross-linking agent. A preferred cross-linking agent forthis process is a carbodiimide, and a particularly preferredcross-linking agent here is the water-soluble carbodiimideN-(3-dimethyl-aminopropyl)-N-ethylcarbodiimide (EDC). In this method,the cross-linking agent is added to an aqueous solution of thepolysaccharide and protein, at an acidic pH and a temperature of about0° C. to 50° C., preferably about 4° C. to 37° C., and allowed to reactfor up to about 48 hrs, preferably up to about 24 hrs. The hydrogel soformed is then isolated, typically by centrifugation, and washed with asuitable solvent to remove uncoupled material.

(2) A mixture of the selected polysaccharide or mucopolysaccharide andprotein is treated with a cross-linking agent having at least twoaldehyde groups, thus forming Schiff-base bonds between the components.These bonds are then reduced with an appropriate reduction agent to givestable carbon-nitrogen bonds. A particularly preferred cross-linkingagent in this procedure is glutaraldehyde, while a particularlypreferred reduction agent is NaCNBH₃. The hydrogel matrix is isolatedand purified as described above.

Prior to cross-linking, if desired, the polysaccharide component, e.g.,heparin, can be partially de-N-sulfated via hydrolysis of N-HSO₃ groupsto increase the number of free amine moieties available forcross-linking.

(3) The carboxylic and/or hydroxyl groups of a polysaccharide ormucopolysaccharide present in quaternary ammonium salt form--e.g., withTriton-B™ are preactivated by treatment with carbonyldiimidazole in anonaqueous medium, e.g., formamide. This is followed by reaction withsaccharine and subsequent reaction with the protein in an aqueousmedium. Reaction time and temperature are the same as in (1) above.

(4) A conjugate of a polysaccharide or a mucopolysaccharide with aprotein may be prepared as described in U.S. Pat. No. 4,526,714 toFeijen et al., the disclosure of which is incorporated by reference. Asdescribed in that patent, conjugates of albumin and heparin may beprepared using EDC as the coupling agent.

The degree of cross-linking in the hydrogel, like the composition of thehydrogel itself, affects the degradation kinetics, loading, and overalltimed release profile of the matrix. That is, a higher degree ofcross-linking will generally result in slower degradation and release,while a lower degree of cross-linking will give faster degradation andrelease.

The hydrogel so formed is loaded with a selected drug by immersion in asolution containing the drug. Typically, hydrogels (albuminmicrospheres, for instance) are loaded by carrying out the crosslinkingprocess in the presence of the drug. Alternatively, some hydrogels areloaded by immersion in a solution of the drug. Alternatively, somehydrogels are loaded by immersion in a solution of the drug in organicsolvent(s), followed by evaporation of the organic solvent(s) afterloading. The hydrogels of the present invention enable one to dispensewith the use of organic solvents, and eliminate the possibility ofcontamination of the hydrogel with organic residues. That is, with thepresent method, the hydrogels may be loaded in an aqueous phase insteadof in an organic solvent. With the present method, the hydrogels(microspheres) may be loaded in an aqueous phase drug solution after thehydrogel has been prepared and purified by washing.

The degree of drug loading is to a large extent dependent on the ionicstrength of the aqueous system. In matrices formed from the ionicallycharged heparin (or analogs) and proteins, the degree of swellingincreases significantly with decreasing ionic strength in thesurrounding medium. Temperature may also be used to vary the degree ofdrug loading, as typically one will obtain a greater degree of drugloading at elevated temperatures due to higher swelling and drugsolubility.

Another variable which influences the degree of drug loading is pH.Depending on the polysaccharide and protein used, changing the pH altersthe degree of ionization, which will affect the swelling behavior of thegel and allow further flexibility in drug loading.

After equilibration, the loaded gels are dried in vacuo under ambientconditions, and stored.

A wide variety of drugs may be incorporated into the hydrogel matrices,including low molecular weight drugs like hormones, cytostatic agentsand antibiotics, peptides and high molecular weight drugs like proteins,enzymes and anticoagulants (such as heparin). Virtually any drug may beloaded into the hydrogel matrices, providing that considerations such assurface charge, size, geometry and hydrophilicity are taken intoaccount. For example, incorporation and release of a high molecularweight drug will typically require a hydrogel having a generally lowerdegree of cross-linking. The release of a charged drug will be stronglyinfluenced by the charge and charge density available in the hydrogel aswell as by the ionic strength of the surrounding media.

The rate of drug release from the matrices can also be influenced bypost-treatment of the hydrogel formulations. For example, heparinconcentration at the hydrogel surface can be increased by reaction ofthe formulated hydrogels with activated heparin (i.e., heparin reactedwith carbonyldiimidazole and saccharine) or with heparin containing onealdehyde group per molecule. A high concentration of heparin at thehydrogel surface will form an extra "barrier" for positively chargeddrugs at physiological pH values. Another way of accomplishing the sameresult is to treat the hydrogels with positively charged macromolecularcompounds like protamine sulfate, polylysine, or like polymers. Still afurther way of varying hydrogel permeability is to treat the surfaceswith biodegradable block copolymers containing hydrophilic andhydrophobic blocks. The hydrophilic block can be a positively chargedpolymer like polylysine (which is able to covalently bind to thenegatively charged heparin), while the hydrophobic block can be abiodegradable poly(α-amino acid) like poly(L-alanine), poly(L-leucine)or similar polymers.

It should be noted that several mechanisms are involved in the rate andextent of drug release. In the case of very high molecular weight drugs,the rate of release will be more dependent on the rate of hydrogelbiodegradation. With lower molecular weight drugs, drug release will bemore dominated by diffusion. In either case, depending on the hydrogelcomponents selected, ionic exchange can also play a major role in theoverall release profile. This is particularly true in applicants'preferred embodiment in which the hydrogel matrices have a substantialdegree of ionic charge, e.g., matrices formed from ionically chargedproteins (e.g., albumin) and heparin analogs.

The hydrogel matrices can be formed into capsules, tablets, films,microspheres, or the like. The compositions formulated using thehydrogel matrices can include conventional pharmaceutical carriers orexcipients, adjuvants, etc. Matrices in the form of discs, slabs orcylinders can be used as implants, while microspheres can be applied assubcutaneous, intramuscular, intravenous or intra-arterial injectables.The size of the hydrogel bodies can be selected so as to direct ultimateplacement. That is, depending on size, intravenously introducedmicrospheres may be physically trapped in the capillary beds of thelungs (size >7 μm), phagocytosed by cells of the RES system (size >100nm) which will locate the particles mainly in the liver and spleen, ormay become lodged at extracellular sites (size <100 nm).

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples which follow are intendedto illustrate and not limit the scope of the invention, which is definedby the scope of the appended claims. Other aspects, advantages andmodifications within the scope of the invention will be apparent tothose skilled in the art to which the invention pertains.

EXAMPLE 1 Preparation of a Biodegradable Hydrogel

Heparin (400 mg, 0.036 mmole) was added to 750 ml double distilledwater. Human serum albumin ("HSA", 550 mg, 0.0085 mmole) was added to1.0 ml double distilled water, and both solutions were kept at 4° C. todissolve overnight. N-(3-dimethylaminopropyl)-N-ethylcarbodiimide("EDC"), 94 mg, was then added to 250 ml double distilled water anddissolved at 4° C. The heparin solution, along with 1 ml of the albuminsolution and a stir bar, was placed in a 2 ml polyethylene-polypropylenesyringe of which the top had been cut off. A plunger was placed on thesyringe and the solutions were thoroughly mixed. The EDC solution wasadded and the mixture was mixed again. All steps were carried out at 4°C.

After 24 hours, the resulting gel was removed from the syringe byswelling the syringe in toluene, and the gel was then equilibrated withphosphate buffered saline ("PBS") to remove uncoupled material.

FIG. 1 outlines the general reaction scheme for this synthesis.

EXAMPLE 2 Preparation of Cross-linked Microspheres

Albumin-heparin microspheres were synthesized according to the reactionscheme shown in FIG. 2. Pure olive oil (250 ml) was added to aflat-bottomed 400 ml beaker. A motor-driven double-bladed stirring barwas then submerged about two-thirds of the way into the oil. Afterstirring the oil for 30 minutes at 1500 rpm, 0.8 ml of an aqueoussolution of albumin and heparin (.sup.˜4:1, w/w) was added to thestirring oil with a 20 gauge syringe. The mixture was then stirred for15 minutes. A solution of EDC in water (112 mgs/ml) was then addeddropwise with a syringe, and the mixture was stirred overnight. Themicrospheres (designated "chemically stabilized" in FIG. 2) wereisolated by centrifuging at 1000 rpm for 10 minutes and weresubsequently vacuum filtered using a Teflon filter (pore size 0.45microns) and washed with ether. The beads were then lyophilized andplaced under vacuum overnight.

Other possibilities for obtaining the albumin-heparin microspheres arealso outlined in FIG. 2. "Denaturation stabilized" microspheres areprepared as described above, except that no cross-linking agent is used."Heat-stabilized" microspheres are also prepared in the absence of across-linking agent, but at a temperature of about 100-170° C.,typically about 130° C.

A modified synthesis scheme for preparinq albumin-heparin microspheres,using a water-in-oil emulsion, is shown in the reaction scheme of FIG.3. In this method, 2.00 g Pluronic F-68 (a trademark of BASF WyandotteCorp., Wyandotte, Mich., for a high molecular weight polyoxyalkyleneether) was dissolved in 8.0 ml CHCl₃ in a 20 ml glass scintillationvial. Albumin (100 mg) and heparin (50.0 mg) were dissolved in 500 μlwater and then added to the surfactant solution to form an emulsion. AnEDC solution (24.0 mg/100μl) was injected into the emulsion and themixture was stirred overnight. Isolation of the microspheres was carriedout as described previously. All steps were carried out at 4° C.

FIGS. 4 and 5 are scanning electron micrographs of albumin-heparin andalbumin microspheres, respectively, which were synthesized according tothe method for preparing "chemically stabilized" microspheres asoutlined above. In the case of the albumin microspheres, the procedureoutlined above was followed except that heparin was omitted.

EXAMPLE 3 Swelling Behavior of Albumin-Heparin Hydrogels

Swelling behavior of the albumin-heparin microspheres prepared as in theprevious example (using varying amounts of heparin) was examined asfollows.

a. The microspheres were placed in PBS buffer solution, pH 7.4, at 22°C., and the uptake of the buffer solution was monitored. As may beconcluded from the graph shown in FIG. 6, uptake of the buffer solutionincreased with heparin content. Thus, to be able to "load" more druginto the hydrogel matrices, heparin content should be correspondinglyincreased.

b. Swelling studies were also carried out in PBS buffer, pH 7.4, atvarying ionic strengths. Equilibrium fractions of solution in thehydrogels were obtained for hydrogels of varying cross-link density.These values are presented in FIGS. 7 and 8. In pure water, where theshielding effects of counterions in solution can't mask the fixedcharges within the hydrogels, swelling occurs until the hydrogel ismechanically very weak. These figures also demonstrate that the amountof "loading" that is possible is also dependent on the amount ofcross-linking agent used as well as on the ionic strength of the solventused.

c. Further swelling studies were done to evaluate the effect of pH. Asabove, the studies were carried out in PBS buffer at 22° C. Here, theionic strength of the solution was maintained at 0.15. As illustrated inFIG. 9, at low pH, the unreacted carboxylic acids (pKa about 4.2) arelargely unionized, thus giving lower swelling. At higher pHs, swellingis correspondingly higher as well. This suggests that amines lose theirprotonation at higher pHs, thus reducing attractive electrostaticinteraction.

EXAMPLE 4 In Vitro Release of a Protein from Hydrogels

Chicken egg albumin (mol. wt. - 45,000) was dissolved in 4° C. in doubledistilled water to make a final 10% (w/v) solution. The gels were thenplaced in 1 ml of these protein solutions for drug loading. Whenequilibrium was attained, the gels were subsequently dried at roomtemperature.

The dried protein loaded discs were then placed in 50 ml (1 disc per 50ml) isotonic PBS buffer, pH=7.40 w/ 0.1% sodium azide at roomtemperature. Samples of buffer solution were withdrawn at variousintervals and assayed for chicken egg albumin. Release was quantified byUV spectroscopy (λmax=279.4 nm), and is shown in FIG. 10.

EXAMPLE 5 Effect of Composition on Macromolecular Release

The effect of albumin/heparin composition on macromolecular release wasevaluated. Aqueous solutions containing either 28.6% and 17.1% albuminand heparin, respectively (i.e., a composition that is 5:3 wt./wt.albumin:heparin) or 34.3% and 11.4% albumin and heparin (i.e., acomposition that is 6:2 wt./wt. albumin: heparin) were prepared. The pHwas adjusted to 5.5 and the solutions cooled to 4° C. EDC was then addedto both solutions to give 7.5% EDC, minimizing the exposure of themixtures to air during EDC addition. The mixtures were then injectedinto film-shaped Mylar® molds which were refrigerated at 4° C. overnightto allow in situ cross-linking of the albumin and heparin. The resultantgels were removed from the molds and discs were cut from the film with acork bore. Final disc dimensions were 12.8 mm in diameter by 1.9 mm inthickness.

Unincorporated albumin and heparin were then exhaustively extracted inisotonic PBS containing 0.1% sodium azide until no extractablecomponents could be detected by UV spectroscopy (for albumin) ortoluidine blue assays (for heparin).

Lysozyme, a 14,400 molecular weight protein, was loaded into thealbumin-heparin gels by solution sorption techniques. Hydrogel discswere immersed in 20 ml of a 0.2% lysozyme aqueous solution, pH 7.3, andequilibrated for 50 hours. The lysozyme-loaded discs were removed fromthe loading solutions, dried with absorbent paper to removesurface-associated lysozyme, and dried at room temperature overnight.

Dried lysozyme-loaded discs were first weighed and then immersed in 50ml (1 disc per 50 ml) PBS containing 0.1% sodium azide. Samples of thePBS were withdrawn at scheduled time points for lysozyme quantitation byUV spectroscopy (280.8 mm). Lysozyme release versus time (t) and t^(1/2)is presented in FIGS. 11 and 12, respectively. From the release data,the lysozyme diffusion coefficients were determined to be 1 82×10⁻⁸ cm²/sec and 9.62×10⁻⁹ cm² /sec for 6:2 w/w and 5:3 w/w albumin-heparinhydrogels, respectively. As expected, then, a higher percentage ofheparin in the hydrogel will decrease the release rate of the trappedpharmacologically active agent, presumably through ionic exchangeinteractions.

What is claimed is:
 1. A method of preparing microspheres useful in thecontrolled release of drugs, comprising:(a) preparing an aqueoussolution of a selected protein and a polysaccharide, wherein the weightratio of polysaccharide to protein in the solution is in the range ofabout 10:90 to 90:10; (b) admixing the protein solution with oil toprovide an emulsion, wherein the volume ratio of oil to protein is inthe range of about 1:1 to 500:1; (c) introducing a cross-linking agentinto the emulsion so as to cross-link the protein and polysaccharide;and (d) isolating the microspheres so formed.
 2. A method of preparingmicrospheres useful in the controlled release of drugs, comprising:(a)preparing an aqueous solution of a selected protein and apolysaccharide, wherein the weight ratio of polysaccharide to protein inthe solution is in the range of about 10:90 to 90:10; (b) introducing across-linking agent into the solution so as to cross-link the proteinand polysaccharide, and to provide a cross-linked mixture; (c) admixingthe cross-linked mixture with oil to provide an emulsion, wherein thevolume ratio of oil to protein is in the range of about 1:1 to 500:1;and
 3. A method of preparing microspheres useful in the controlledrelease of drugs, comprising:preparing an aqueous solution of a selectedprotein and a polysaccharide, wherein the weight ratio of polysaccharideto protein in the solution is in the range of about 10:90 to 90:10; (b)admixing the aqueous solution with oil to provide an emulsion, whereinthe volume ratio of oil to protein is in the range of about 1:1 to500:1; (c) isolating the microspheres so formed; and (d) heating themicrospheres to a temperature in the range of about 100° C. to about170° C.
 4. The method of any one of claims 1, 2 or 3, further comprisingloading an effective amount of a drug into the microspheres by immersingthe microspheres in a solution of the drug.
 5. The method of claim 4wherein the drug is selected from the group consisting of proteins,enzymes, mucopolysaccharides, peptides, hormones, antibodies andcytostatic agents.
 6. The method of any one of claims 1, 2 or 4, whereinthe polysaccharide is selected from the group consisting of heparin,heparin fragments, heparan, heparan sulfate, chondroitin sulfate, andmixtures thereof.
 7. The method of claim 6, wherein the polysaccharideis selected from the group consisting of heparin, heparin fragments,heparan and heparan sulfate.
 8. The method of any one of claims 1, 2 or3, wherein the protein is selected from the group consisting of albumin,casein, fibrinogen, γ-globulin, hemoglobin, ferritin, elastin andsynthetic α-amino peptides.
 9. The method of claim 8 wherein the proteinis albumin.
 10. The method of either of claims 1 or 2, wherein thecross-linking agent is an amide bond-forming agent.
 11. The method ofclaim 10, wherein the amide-bond forming agent is a carbodiimide. 12.The method of claim 11, wherein the carbodiimide isN-(3-dimethylaminopropyl)-N'-ethylcarbodiimide.
 13. Microspheresproduced by the method of claim
 1. 14. Microspheres produced by themethod of claim
 2. 15. Microspheres produced by the method of claim 3.16. Drug-containing microspheres produced by the method of claim
 4. 17.Drug-containing microspheres produced by the method of claim 3.